Kristoffer Valerie

Virginia Commonwealth University, Richmond, VA, United States

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Publications (81)464.18 Total impact

  • Jason M Beckta, Syed Farhan Ahmad, Hu Yang, Kristoffer Valerie
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    ABSTRACT: Despite intense studies, highly effective therapeutic strategies against cancer have not yet been fully exploited, because few true cancer-specific targets have been identified. Most modalities, perhaps with the exception of radiation therapy, target proliferating cells, which are also abundant in normal tissues. Thus, most current cancer treatments have significant side effects. More than 10 years ago, the tumor suppressor p53 was first explored as a cancer-specific target. At the time, the approach was to introduce a normal p53 gene into mutant p53 (mp53) tumor cells to induce cell cycle arrest and apoptosis. However, this strategy did not hold up and mostly failed in subsequent clinical studies. Recent research developments have now returned p53 to the limelight. Several studies have reported that mutant or null p53 tumor cells undergo apoptosis more easily than genetically matched, normal p53 counterparts when inhibiting a specific stress kinase in combination with standard chemotherapy or when exposed to an ataxia-telangiectasia mutated (ATM) kinase inhibitor and radiation, thus achieving true cancer specificity in animal tumor models. This short review highlights several of these recent studies, discusses possible mechanism(s) for mp53-mediated "synthetic lethality," and the implications for cancer therapy.
    Cell cycle (Georgetown, Tex.) 02/2014; 13(5). · 5.24 Impact Factor
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    ABSTRACT: To track the processing of damaged DNA double-strand break (DSB) ends in vivo, a method was devised for quantitative measurement of 3'-phosphoglycolate (PG) termini on DSBs induced by the non-protein chromophore of neocarzinostatin (NCS-C) in the human Alu repeat. Following exposure of cells to NCS-C, DNA was isolated, and labile lesions were chemically stabilized. All 3'-phosphate and 3'-hydroxyl ends were enzymatically capped with dideoxy termini, whereas 3'-PG ends were rendered ligatable, linked to an anchor, and quantified by real-time Taqman polymerase chain reaction. Using this assay and variations thereof, 3'-PG and 3'-phosphate termini on 1-base 3' overhangs of NCS-C-induced DSBs were readily detected in DNA from the treated lymphoblastoid cells, and both were largely eliminated from cellular DNA within 1 h. However, the 3'-PG termini were processed more slowly than 3'-phosphate termini, and were more persistent in tyrosyl-DNA phosphodiesterase 1-mutant SCAN1 than in normal cells, suggesting a significant role for tyrosyl-DNA phosphodiesterase 1 in removing 3'-PG blocking groups for DSB repair. DSBs with 3'-hydroxyl termini, which are not directly induced by NCS-C, were formed rapidly in cells, and largely eliminated by further processing within 1 h, both in Alu repeats and in heterochromatic α-satellite DNA. Moreover, absence of DNA-PK in M059J cells appeared to accelerate resolution of 3'-PG ends.
    Nucleic Acids Research 12/2013; · 8.28 Impact Factor
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    ABSTRACT: Despite similar structures and DNA binding profiles, two recently synthesized dinuclear platinum compounds are shown to elicit highly divergent effects on cell cycle progression. In colorectal HCT116 cells, BBR3610 shows a classical G2/M arrest with initial accumulation in S phase, but the derivative compound BBR3610-DACH, formed by introduction of the 1,2-diaminocyclohexane (DACH) as carrier ligand, results in severe G1/S as well as G2/M phase arrest, with nearly complete S phase depletion. The origin of this unique effect was studied. Cellular interstrand crosslinking as assayed by comet analysis was similar for both compounds, confirming previous in vitro results obtained on plasmid DNA. Immunoblotting revealed a stabilization of p53 and concomitant transient increases in p21 and p27 proteins after treatment with BBR3610-DACH. Cell viability assays and cytometric analysis of p53 and p21 null cells indicated that BBR3610-DACH-induced cell cycle arrest was p21-dependent and partially p53-dependent. However, an increase in the levels of cyclin E was observed with steady state levels of CDK2 and Cdc25A, suggesting that the G1 block occurs downstream of CDK/cyclin complex formation. The G2/M block was corroborated with decreased levels of cyclin A and cyclin B1. Surprisingly, BBR3610-DACH-induced G1 block was independent of ATM and ATR. Finally, both compounds induced apoptosis, with BBR3610-DACH showing a robust PARP-1 cleavage that was not associated with caspase-3/7 cleavage. In summary, BBR3610-DACH is a DNA binding platinum agent with unique inhibitory effects on cell cycle progression that could be further developed as a chemotherapeutic agent complementary to cisplatin and oxaliplatin.
    Biochemical pharmacology 10/2013; · 4.25 Impact Factor
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    ABSTRACT: Stat3 is a key mediator in the development of many cancers. For 20 years it has been assumed that Stat3 mediates its biological activities as a nuclear localized transcription factor activated by many cytokines. However, recent studies from this lab and others indicate that Stat3 has an independent function in the mitochondria (mitoStat3) where it controls the activity of the electron transport chain (ETC) and mediates Ras induced transformation of mouse mebryo fibroblasts (MEFs). The actions of mitoStat3 in controlling respiration and Ras transformation are mediated by the phosphorylation state of serine 727. To address the role of mitoStat3 in the pathogenesis of cells that are transformed we used 4T1 breast cancer cells which form tumors that metastasize in immunocompetent mice. Substitution of Ser727 for an alanine or aspartate in Stat3 that has a mitochondrial localization sequence, (MLS-Stat3), has profound effects on tumor growth, complex I activity of the ETC and accumulation of reactive oxygen species. Cells expressing MLS-Stat3(S727A) display slower tumor growth, decreased complex I activity of the ETC and increased ROS accumulation under hypoxia compared with cells expressing MLS-Stat3. In contrast, cells expressing MLS-Stat3(S727D) show enhanced tumor growth, and complex I activity and decreased production of ROS. These results highlight the importance of serine 727 of mitoStat3 in breast cancer and suggest a novel role for mitoStat3 in regulation of ROS concentrations through its action on the ETC.
    Journal of Biological Chemistry 09/2013; · 4.65 Impact Factor
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    ABSTRACT: PURPOSE: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer with a median survival of only 12-15 months. Current standard treatment consists of surgery followed by chemoradiation. The poor survival of GBM patients is due to aggressive tumor invasiveness, an inability to remove all tumor tissue, and an innate tumor chemo- and radioresistance. ATM, ataxia telangiectasia (A-T) mutated, is an excellent target for radiosensitizing GBM because of its critical role in regulating the DNA damage response and p53, among other cellular processes. As a first step toward this goal, we recently showed that the novel ATM kinase inhibitor KU-60019 reduced migration, invasion, growth, and potently radiosensitized human glioma cells in vitro. EXPERIMENTAL DESIGN: Using orthotopic xenograft models of GBM, we now show that KU-60019 is also an effective radiosensitizer in vivo. Human glioma cells expressing reporter genes for monitoring tumor growth and dispersal were grown intra-cranially, and KU-60019 was administered intra-tumorally by convection-enhanced delivery or osmotic pump. RESULTS: Our results demonstrate that the combined effect of KU-60019 and radiation significantly increased survival of mice 2-3 fold over controls. Importantly, we show that glioma with mutant p53 is much more sensitive to KU-60019 radiosensitization than genetically matched wild-type glioma. CONCLUSIONS: Taken together, our results suggest that an ATM kinase inhibitor may be an effective radiosensitizer and adjuvant therapy for patients with mutant p53 brain cancers.
    Clinical Cancer Research 04/2013; · 7.84 Impact Factor
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    ABSTRACT: Bioluminescence imaging (BLI) is a relatively new noninvasive technology used for quantitative assessment of tumor growth and therapeutic effect in living animal models. BLI involves the generation of light by luciferase-expressing cells following administration of the substrate luciferin in the presence of oxygen and ATP. In the present study, the effects of hypoxia, hypoperfusion, and pH on BLI signal (BLS) intensity were evaluated in vitro using cultured cells and in vivo using a xenograft model in nude mice. The intensity of the BLS was significantly reduced in the presence of acute and chronic hypoxia. Changes in cell density, viability, and pH also affected BLS. Although BLI is a convenient non-invasive tool for tumor assessment, these factors should be considered when interpreting BLS intensity, especially in solid tumors that could be hypoxic due to rapid growth, inadequate blood supply, and/or treatment.
    International journal of molecular imaging. 01/2013; 2013:287697.
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    ABSTRACT: Water-soluble camptothecin (CPT)-polyoxetane conjugates were synthesized using a clickable polymeric platform P(EAMO) that was made by polymerization of acetylene-functionalized 3-ethyl-3-(hydroxymethyl)oxetane (i.e., EAMO). CPT was first modified with a linker 6-azidohexanoic acid via an ester linkage to yield CPT-azide. CPT-azide was then click coupled to P(EAMO) in dichloromethane using bromotris(triphenylphosphine)copper(I)/N,N-diisopropylethylamine. For water solubility and cytocompatibility improvement, methoxypolyethylene glycol azide (mPEG-azide) was synthesized from mPEG 750 g mol(-1) and click grafted using copper(II) sulfate and sodium ascorbate to P(EAMO)-g-CPT. (1)H NMR spectroscopy confirmed synthesis of all intermediates and the final product P(EAMO)-g-CPT/PEG. CPT was found to retain its therapeutically active lactone form. The resulting P(EAMO)-g-CPT/PEG conjugates were water-soluble and produced dose-dependent cytotoxicity to human glioma cells and increased γ-H2AX foci formation, indicating extensive cell cycle-dependent DNA damage. Altogether, we have synthesized CPT-polymer conjugates able to induce controlled toxicity to human cancer cells.
    Molecular Pharmaceutics 10/2012; · 4.57 Impact Factor
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    ABSTRACT: Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.
    Cell cycle (Georgetown, Tex.) 03/2012; 11(6):1167-73. · 5.24 Impact Factor
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    ABSTRACT: Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitinligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.
    Cell cycle (Georgetown, Tex.) 02/2012; 11(4):687-94. · 5.24 Impact Factor
  • Jason M Beckta, Scott C Henderson, Kristoffer Valerie
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    ABSTRACT: Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations(1). To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in(2)). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1. The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones(3,4), forming foci within 5-15 minutes(5). The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair(6). Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins(7-9). Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs(6). DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis(10). In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs(11). In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis(12). Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.
    Journal of Visualized Experiments 01/2012;
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    ABSTRACT: Research into the diagnosis and treatment of central nervous system (CNS) diseases has been enhanced by rapid advances in nanotechnology and an expansion in the library of nanostructured carriers. This review discusses the latest applications of nanomaterials in the CNS with an emphasis on brain tumors. Novel administration routes and transport mechanisms for nanomaterial-mediated CNS delivery of diagnostic and therapeutic agents to bypass or cross the blood brain barrier (BBB) are also discussed. These include temporary disruption of the BBB, use of impregnated polymers (polymer wafers), convection-enhanced delivery (CED), and intranasal delivery. Moreover, an in vitro BBB model capable of mimicking geometrical, cellular and rheological features of the human cerebrovasculature has been developed. This is a useful tool that can be used for screening CNS nanoparticles or therapeutics prior to in vivo and clinical investigation. A discussion of this novel model is included.
    Advanced drug delivery reviews 12/2011; 64(7):605-13. · 11.96 Impact Factor
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    Sarah E Golding, Kristoffer Valerie
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    ABSTRACT: Comment on: Fraser, M, et al. Cell Cycle 2011; 10:2218-32.
    Cell cycle (Georgetown, Tex.) 10/2011; 10(19):3227. · 5.24 Impact Factor
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    ABSTRACT: Radiation and other types of DNA damaging agents induce a plethora of signaling events simultaneously originating from the nucleus, cytoplasm, and plasma membrane. As a result, this presents a dilemma when seeking to determine causal relationships and better insight into the intricacies of stress signaling. ATM plays critical roles in both nuclear and cytoplasmic signaling, of which, the DNA damage response (DDR) is the best characterized. We have recently created experimental conditions where the DNA damage signal alone can be studied while minimizing the influence from the extranuclear compartment. We have been able to document pro-survival and growth promoting signaling (via ATM-AKT-ERK) resulting from low levels of DSBs (equivalent to ≤2 Gy). More extensive DSBs (>2 Gy eq.) result in phosphatase-mediated ERK dephosphorylation, and thus shutdown of ERK signaling. In contrast, radiation does not result in such dephosphorylation even at very high doses. We propose that phosphatases are inactivated perhaps as a result of reactive oxygen species, which does not occur in response to 'pure' DNA damage. Our findings suggest that clinically relevant radiation doses, which are intended to halt tumor growth and induce cell death, are unable to inhibit tumor pro-survival signaling via ERK dephosphorylation.
    Radiotherapy and Oncology 07/2011; 101(1):13-7. · 4.52 Impact Factor
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    ABSTRACT: We introduced a K1702M mutation in the BRCA1 BRCT domain known to prevent the binding of proteins harboring pS-X-X-F motifs such as Abraxas-RAP80, BRIP1, and CtIP. Surprisingly, rather than impairing homologous recombination repair (HRR), expression of K1702M resulted in hyper-recombination coinciding with an accumulation of cells in S-G2 and no effect on nonhomologous end-joining. These cells also showed increased RAD51 and RPA nuclear staining. More pronounced effects were seen with a naturally occurring BRCT mutant (M1775R) that also produced elevated levels of ssDNA, in part co-localizing with RPA, in line with excessive DNA resection. M1775R induced unusual, thread-like promyelocytic leukemia (PML) nuclear bodies and clustered RPA foci rather than the typical juxtaposed RPA-PML foci seen with wild-type BRCA1. Interestingly, K1702M hyper-recombination diminished with a second mutation in the BRCA1 RING domain (I26A) known to reduce BRCA1 ubiquitin-ligase activity. Thesein vitro findings correlated with elevated nuclear RAD51 and RPA staining of breast cancer tissue from a patient with the M1775R mutation. Altogether, the disruption of BRCA1 (BRCT)-pS-X-X-F protein binding results in ubiquitination-dependent hyper-recombination via excessive DNA resection and the appearance of atypical PML-NBs. Thus, certain BRCA1 mutations that cause hyper-recombination instead of reduced DSB repair might lead to breast cancer.
    Aging 05/2011; 3(5):515-32. · 4.70 Impact Factor
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    ABSTRACT: Ionizing radiation (IR) triggers many signaling pathways primarily originating from either damaged DNA or non-nuclear sources such as growth factor receptors. Thus, to study the DNA damage-induced signaling component alone by irradiation would be a challenge. To generate DNA double-strand breaks (DSBs) and minimize non-nuclear signaling, human cancer cells having bromodeoxyuridine (BrdU) - substituted DNA were treated with the photosensitizer Hoechst 33258 followed by long wavelength UV (UV-A) treatment (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose- dependent generation of DSBs equivalent to radiation doses between 0.2 - 20 Gy, as determined by pulsed-field gel electrophoresis, and accompanied by dose-dependent ATM (ser-1981), H2AX (ser-139), Chk2 (thr-68), and p53 (ser-15) phosphorylation. Interestingly, low levels (≤ 2 Gy equivalents) of BrdU photolysis stimulated ERK phosphorylation whereas higher (> 2 Gy eq.) resulted in ERK dephosphorylation. ERK phosphorylation was ATM-dependent whereas dephosphorylation was ATM-independent. The ATM-dependent increase in ERK phosphorylation was also seen when DSBs were generated by transfection of cells with an EcoRI expression plasmid or by electroporation of EcoRI enzyme. Furthermore, AKT was critical for transmitting the DSB signal to ERK. Altogether, our results show that low levels of DSBs trigger ATM- and AKT-dependent ERK pro-survival signaling and increased cell proliferation whereas higher levels result in ERK dephosphorylation consistent with a dose-dependent switch from pro-survival to anti-survival signaling.
    Cell cycle (Georgetown, Tex.) 02/2011; 10(3):481-91. · 5.24 Impact Factor
  • Fuel and Energy Abstracts 01/2011; 81(2).
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    ABSTRACT: We recently demonstrated that human embryonic stem cells (hESCs) utilize homologous recombination repair (HRR) as primary means of double-strand break (DSB) repair. We now show that hESCs also use nonhomologous end joining (NHEJ). NHEJ kinetics were several-fold slower in hESCs and neural progenitors (NPs) than in astrocytes derived from hESCs. ATM and DNA-PKcs inhibitors were ineffective or partially effective, respectively, at inhibiting NHEJ in hESCs, whereas progressively more inhibition was seen in NPs and astrocytes. The lack of any major involvement of DNA-PKcs in NHEJ in hESCs was supported by siRNA-mediated DNA-PKcs knockdown. Expression of a truncated XRCC4 decoy or XRCC4 knock-down reduced NHEJ by more than half suggesting that repair is primarily canonical NHEJ. Poly(ADP-ribose) polymerase (PARP) was dispensable for NHEJ suggesting that repair is largely independent of backup NHEJ. Furthermore, as hESCs differentiated a progressive decrease in the accuracy of NHEJ was observed. Altogether, we conclude that NHEJ in hESCs is largely independent of ATM, DNA-PKcs, and PARP but dependent on XRCC4 with repair fidelity several-fold greater than in astrocytes.
    Aging 09/2010; 2(9):582-96. · 4.70 Impact Factor
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    Bret R Adams, Sarah E Golding, Raj R Rao, Kristoffer Valerie
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    ABSTRACT: The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.
    PLoS ONE 01/2010; 5(4):e10001. · 3.73 Impact Factor
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    ABSTRACT: Ataxia telangiectasia (A-T) mutated (ATM) is critical for cell cycle checkpoints and DNA repair. Thus, specific small molecule inhibitors targeting ATM could perhaps be developed into efficient radiosensitizers. Recently, a specific inhibitor of the ATM kinase, KU-55933, was shown to radiosensitize human cancer cells. Herein, we report on an improved analogue of KU-55933 (KU-60019) with K(i) and IC(50) values half of those of KU-55933. KU-60019 is 10-fold more effective than KU-55933 at blocking radiation-induced phosphorylation of key ATM targets in human glioma cells. As expected, KU-60019 is a highly effective radiosensitizer of human glioma cells. A-T fibroblasts were not radiosensitized by KU-60019, strongly suggesting that the ATM kinase is specifically targeted. Furthermore, KU-60019 reduced basal S473 AKT phosphorylation, suggesting that the ATM kinase might regulate a protein phosphatase acting on AKT. In line with this finding, the effect of KU-60019 on AKT phosphorylation was countered by low levels of okadaic acid, a phosphatase inhibitor, and A-T cells were impaired in S473 AKT phosphorylation in response to radiation and insulin and unresponsive to KU-60019. We also show that KU-60019 inhibits glioma cell migration and invasion in vitro, suggesting that glioma growth and motility might be controlled by ATM via AKT. Inhibitors of MEK and AKT did not further radiosensitize cells treated with KU-60019, supporting the idea that KU-60019 interferes with prosurvival signaling separate from its radiosensitizing properties. Altogether, KU-60019 inhibits the DNA damage response, reduces AKT phosphorylation and prosurvival signaling, inhibits migration and invasion, and effectively radiosensitizes human glioma cells.
    Molecular Cancer Therapeutics 10/2009; 8(10):2894-902. · 5.60 Impact Factor
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    ABSTRACT: Although tyrosyl-DNA phosphodiesterase (TDP1) is capable of removing blocked 3' termini from DNA double-strand break ends, it is uncertain whether this activity plays a role in double-strand break repair. To address this question, affinity-tagged TDP1 was overexpressed in human cells and purified, and its interactions with end joining proteins were assessed. Ku and DNA-PKcs inhibited TDP1-mediated processing of 3'-phosphoglycolate double-strand break termini, and in the absence of ATP, ends sequestered by Ku plus DNA-PKcs were completely refractory to TDP1. Addition of ATP restored TDP1-mediated end processing, presumably due to DNA-PK-catalyzed phosphorylation. Mutations in the 2609-2647 Ser/Thr phosphorylation cluster of DNA-PKcs only modestly affected such processing, suggesting that phosphorylation at other sites was important for rendering DNA ends accessible to TDP1. In human nuclear extracts, about 30% of PG termini were removed within a few hours despite very high concentrations of Ku and DNA-PKcs. Most such removal was blocked by the DNA-PK inhibitor KU-57788, but approximately 5% of PG termini were removed in the first few minutes of incubation even in extracts preincubated with inhibitor. The results suggest that despite an apparent lack of specific recruitment of TDP1 by DNA-PK, TDP1 can gain access to and can process blocked 3' termini of double-strand breaks before ends are fully sequestered by DNA-PK, as well as at a later stage after DNA-PK autophosphorylation. Following cell treatment with calicheamicin, which specifically induces double-strand breaks with protruding 3'-PG termini, TDP1-mutant SCAN1 (spinocerebellar ataxia with axonal neuropathy) cells exhibited a much higher incidence of dicentric chromosomes, as well as higher incidence of chromosome breaks and micronuclei, than normal cells. This chromosomal hypersensitivity, as well as a small but reproducible enhancement of calicheamicin cytotoxicity following siRNA-mediated TDP1 knockdown, suggests a role for TDP1 in repair of 3'-PG double-strand breaks in vivo.
    DNA repair 07/2009; 8(8):901-11. · 4.20 Impact Factor

Publication Stats

3k Citations
464.18 Total Impact Points

Institutions

  • 2002–2013
    • Virginia Commonwealth University
      • • Department of Radiation Oncology
      • • Department of Human and Molecular Genetics
      • • Department of Pharmacology and Toxicology
      • • Department of Neurology
      Richmond, VA, United States
  • 2008
    • Lawrence Berkeley National Laboratory
      • Life Sciences Division
      Berkeley, CA, United States
  • 2002–2008
    • Columbia University
      • College of Physicians and Surgeons
      New York City, NY, United States
  • 2003
    • Heinrich-Heine-Universität Düsseldorf
      Düsseldorf, North Rhine-Westphalia, Germany